Some biological, biochemical and morphological changes and their correlations in selected BCG vaccine strains

Some biological, biochemical and morphological changes and their correlations in selected BCG vaccine strains

ffournal of Biological ~tandardizat~n 1976 4, 319--327 S o m e bi morphol co elatio cal, biochemic and al ch ges and their in selected B C G vaccine...

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ffournal of Biological ~tandardizat~n 1976 4, 319--327

S o m e bi morphol co elatio

cal, biochemic and al ch ges and their in selected B C G vaccine

.7. Pr:tchov~,t .~. 2d~ra,$ H . ~/loheIsk~,§ Z. ~gr§ and ff. Galliovd§

Changes m the biolo~caI, bloehem_i~ and morphological properties of three BeG s~ns, C o p e n h a g e n B C G 1331 a n d ~e¢o o f its d ~ v a t l v e s , P r a b ~ B C G 725 S H a n d P r a h a B C G 725 S A S , w e r e s t u d i e d a n d . T h e s e c h a n g e s w e r e c a u s e d b y c h e m i c a l (cult~.Ire medga) and p h y s i ~ factors (fre~z~-drying). T h e highest i.m_rnvJaizingl~t~ncy ~'~d the Im'ghest lipid content were displayed, both prior to and after ~reeze ch-ying, by the Da~_ish s~. cultivation of the Danish str-,dnon different media fin Prague resulted in a d e c r e a s e o f its p o t e n c y a n d lipid c o n t e n t . B o t h o f t h e C z e c h o s l o v a k s t r a i n s d e r i v e d in this m a n n e r , B C G 725 S H a n d B C G 725 S A S , d i f f e r e d m u t u a l l y b y t h e / r L m m t m l z i n g p o t e n c y , llpid c o n t e n t a n d o f L n t a ~ c~LLs. T h e i r r ' ~ a n i z i n g p o t e n c y a n d l i p i d c o n t e n t o f t h e s e t w o s t r a i n s w e r e m ~ v e r s e p r o p o r t i o n ; so w e r e t h e irnrnmnlzing potency and percenta~ of inta~ ria. The expe~mts s u g g e s t e d t h a t s u b s t a n t i a l c h a n g e s d e v e l o p a f t e r p h y s i c a l a c t i o n (freezed-~J;mg) only in stre~as being tx-~q_int~ed on f i ~ cultttre m e ~ F r ~ z e ~ r l n g changed the relationship betwedn Lng p o t e n ~ s-~d Hpld content into a direct one in both of the Czechoslovak straJ~ns. T~ne re~,ionship betweext ~ t m A z flag potenc~l and percentage of Lstact mycobactefia remained inverse. In B e G 72$ S H , total Hpids a n d L t r m a t m ~ g p o t e n c y a n d t h e percem-tag¢ o f k-~tact cells i n c r e a s e d a~er__~-eeze d r y i n g . I ~ B C G 725 S A S o n t h e o t h e r h a t t d t h e i r m x a u n ~ g poten~ fincreased, llpids z e m ~ e d o n a s t a b l e level a n d t h e o f L-tract cells dec~-~ased. T h e substant/al changes in t h e s e p induced by" Jfireeze-dt~Jng s u b s e q u e n t l y r e t u r n e d to t h e originaI v a l u e s i r r e s p e c t i v e o f w h e t h e r t h e strai_jxs h a d u n d e r g o n e a n u m b e r o f freeze-dr~_.ng p r o c e d u r e s o r h a d b e e n p a s s a g e d d i r e c t l y a f t e r a sfingle freeze-d-_~'ing v a n . Aza essential effect w a s t h u s o n l y p r o d u c e d b y t h e first f r e - e z e - ~ g , a n d t h e e.h~sagea ap//ear n o t t o b e stable. * R e c e i v e d f o r p u b l i c a t i o n 20 A p ~ 1976. f S t a t e I r ~ t i t u t e f o r D r u g C o n t r o l ( D i r . J; B~CL~ek, M . D . C . S c . ) , P r a g u e . ! s d H c r o b i o l o # M I n s t i t u t e , }J~_dlcal FacuIg¢, C h a r l e s U n i v e r s i W ( H e a d A s s i s t a n t P r o f e s s o r John, M.D.C.Sc.) Prague. § Institaate o f H y g i e n e a n d E p i (Dh-. P r o f e s s o r F . J a n d a , M . D . D . S c . ) , P r a g u e .

319

J . P R UoC H O

V

A, M . M A R A

H. I'vIOHELSKA, Z. ~IR A N D

J. G A L L I O V A

INTRODUCTION Although there has been a considerable decrease in t u ~ r c u l o s i s morbidity in Czechoslovakia, ha p a r t i c u l ~ among chiIdren and adolescents, prevention of a disease o f such consequence is still indispensable. T h e most i m p o r t a n t component of T B prevention is B C G vaccination. T h e original French B C G sti~in has, in the course of its long-term maintenance u n d e r varying cultivation conditions in different laboratories, undergone some substantial biological, b i o c h e m i ~ and m o r p h o l o ~ c a l changes. T h e selection of a variant of the strain and cultivation s that w ~ r a n t standard properties and t~igh vaccine effectiveness thus becomes a primary problem. Ac ~ e present steady was especially mined at investigating chmnges in the b i o l o g i ~ properties o f vaccine sh-ains of which the most important is Lrnmmaizing potency. AdditionaI aims were to explore, in a model experiment, w h e t h e r there were any concrete relations between the changes concerned (biological, biochemical and morphological) and to estimate the changes quantitatively. MATERIAL

AND

METHODS

Strains and cultivation Danish vaccine prepared from strain B C G 1331 was used as a standard. T h i s strain was pa~saged in Japan on bile-potato, then on asparagine-contaLaing original Sauton's m e d i u m and t h e n freeze-dried. On the basis of our l o n g - t e ~ experiences with this standard strain, the calculated percentage of immunizing potency o f the other s t r ~ s was related statistically 4o its Lrrm~unizing potency. T h e actual study was concerned with the Czechoslovak vaccine strah~ B C G 725, which h a d been derived from batch 725 of the Danish s ~ a i n in 1946. Since 1947 it has been passaged first on Sauton's potato and then d~ectly in Sauton's m e d i u m in which asparagine, has been replaced by e n ~ S c hydrolysate o f easeLn (SH medium). I n addition, the same B C G 725 strain was also studied after maintenance on Sauton's potato and cultivation in o r i ~ n a l Sauton's m e d i u m (SAS m e , u r n ) , containLng ~ p a r a g i n e as the only amino acid.

Freeze-drying of BCG-strab,.s Freeze-dr-ying was performed on an L Z 9B apparatus (Frigera, Kolin, Czechoslovakia). ~ e oalt~are m e d i u m of 72day ~ I t u r e s was remoo~d in a Birghaug b a ~ e r i a l press. T h e bacterial mass was homogenized by shaking with stainless steel balls and diI * U ted with 1~o Na.glutamate to a basic sus1~znsion of 50 m g o f ~ m i d r y wt./mL T h i s suspension was further diluted with 1 % Na-glutmmate to 10 mg[m_l, d i s p e n ~ d in 0.5 ml p o ~ o n s into a m p u l e s and f r o w n in a deep freezer t o - - 4 0 °C for 3 h. T h e anapoules were then transferred into the freeze-drying apparat~as and freez~e-dried for 16-18 h. Using a semiautomatic Edwards appliance, ampoule necks were con~_ricted, and d ~ n g was completed on an Edwards secondary freeze-drier (16 h); ampoules were seaIed u n d e r vacuum.

A

t of model experiment

A B C G stnLLn that had n o t previously been f r e e ~ - d r i e d (Former) was (I) freeze-dried or ( 2 ) s u b j e c t e d to further p ~ a g i n g . Tile freeze-dried strain was seeded and again either (1) pa~aged or (2) freeze-dried r e p u t e d l y (Fig. 1). I n this way three lines were obtained: 320

CHANGES I N B C G

VACCINE STRAINS

(1) non-freeze-dried strain; (2) ~ n freeze-dried once; (3) strain f r e e ~ - d r i e d repeatedly. Since liquid vaccine prepared from non-freeze-dried B C G strain had to be used for guinea pig vaccination in the immunization experLment, none of the Iq-eeze-dried passages were acLm~stered in the ck~¢ form b u t were seeded and only the third to s ~ t h p ~ a g e was employed for liquid-vaccine production and all other investigations. Seeding on Souton~spototo " ~ 0

Singles possoges on Souton's medium

Freeze- drying

j

_

Fig. 1. Arr~'ageme~t o f m o d e l ~ e r ; . m e n t .

Assay of BCG strain immunimhg p o ~ ~ e in.aramizing potency of a strain was estimated on g-ain~ pigs in t e ~

of spleen weight. I t was agsumed that a guinea pig group solely imnaunized but not challenged represented a group given a vaccine of 100% efficacy. On the other'hand, a group solely infected but not d represented a group given an ineffective vaccine, i.e. one of 0% efficacy. Groups of guinea pigs were irranunized with the vaccines u n d e r study and after 1 month were challenged -with ~ I e n t erium tuberculo.~is, strain H 3 7 R v . Six wealds later the animals were killed and their spleens weighed. Mean spleen weights in the individual groups were evaluated statistically (Predc_hov~ & Hejdov~, 1971 ; Prbchov~i & gfr, 1972).

T o ~ lipids were determined via 48-h extraction of washed d ~ bacterial mass ~ t h hot 96% ethanol L'a a Soxhlet apparatus (hq~ra, Galliov~ & Hejdov~, 1971). T h e lipid content was e x p r e ~ e d (a) as percentage of bacterial ~ weight and Co) as percentage of a standard rrfixed-batch sample of Czechoslovak B C G stredn 725 S H ext,racted in parallel Protein assay was performed by determining total mtrogen by the KjeIdahl method and multiplying the values by a factor of 6.25. Polysao:harides were determined by the orcinol method after 21-day e~,'action of dry batten'a! mass ~ t h cold 8~/g if'CA (M~ra et aL, 1971).

NIorphoIogT was examined e l e c t r o n ~ c r o s c o p i ~ l y and the percentage of intact ceils was related to damaged cells. All ~ p l ~ were prepared directly from culture excludLng mechanical homogenization and using two t e c h n i q u ~ in order to ensure at least par'dal verification of results, as follow: 321

] . P R O C H O V A , M . l~i~.RA, H . ~ I O H E L S I ~ . ~ , Z. ~ f R A N D J. G A L L I O V ~ . (a) T h e ultrathin section technique of Ryter, K e l l e n b e ~ r , Birch-Anderson & l'viaaloe (1958) as modified by Mohelsk~f & Prllc~ov~ (1972) was supplemented by a prepolymerization step in pure Vestopal with 1% of activator and 1% of initiator at 4-9 °C for 24 h. Sections were cut on a 1 L K B Ultratome, contrasted with a 0-5% water solution o £ u r a n y I acetate £or 5 man and then ~ t h lead dt.~ate for 2-5 rain ( R e ~ o l d s , 1963). (b) T h e technique of whole specLmens repeatedly washed after fLxation with a 1% solution of O s O 4 in acetate--veronal buffer ~ e r Michaelis (Ryter et aL, 1958) with s~ine mad distilled water. T h e specimens were then applied to a foil and shaded with gold palladium at an angle of 20 °. A PbJlips E M 300 electron microscope was employed: acceleration voltage 80 kV, direct m a g n i f i ~ f i o n × 3 3 000 for u l t , r a ~ sections, × 12 500 for whole specimens. T h e criteria of 'pathologic~ d ~ a g e ' proposed for mycobacteria by A r m s t r o n g & d ' ~ r c y H a r t (1971) were used to expres~s major deviations from n o ~ I bacterial stracture. RESULTS T h e immunizing potency of strain B C G 725.SH decreased ~ e r freeze-drying ~ d subsequently gradually returned to noL~ial values both if the strain was passaged after one freezc-dr-~m4g run only (Fig. 2) or had been freeze-dried repeatedly (Fig. 3). T h e ing potency o f non-freeze-dried B C G 725 S H remained p r a c t i ~ l y unchanged L-~ the course of the experh-nent. immunization pofency Lipids recoicufoted to standard Upids

Percentage of intact ce! Is

r2C

IOC 8C 60 40

0

F

LI

Lx

Lz

Fig. 2. T h e c o m p a r i s o n o f ~ t m i z a t J o n p o t e n c y , lipid c o n t ~ t a n d n u m ~ r o f intact ceils Ln P r a g u e B C G s t ~ c~tiv~ted on SH m~um b e f o r e a n d after free~e-drying.

I n the b i o c h e ~ respect, B C G 725 S H displayed a significant d e c r e e o f the total lipid content ~ e r freeze-drying (Fig. 2). L a t e r in the course o f the experiment the t o ~ Hpids returned to the origh'~al values, similarly as did the in'LmtLnizkng potency, Lrrespective of whether t h e Z ~ r ~ had been freeze-dried once o r u p to four times (Fig. 3). T o ~ proteins, as c a l ~ l a t e d from t o ~ nitrogen a n d t o ~ ~lysaccharidez, displayed i n s i g a ~ t changes in both e x p e r i m e n ~ variants. I n electronmicroscopic representation, the perc~,m~e of intact m y c o b a c t e ~ in a 7-day culture of strain B C G 725 S H rose s u b s t ~ t i ~ l y after the first freeze-drylng (Fig, 2). W h e n freeze-dried once and s u b s e q u e n d y passaged, the s t r ~ exhibited a steep initial

322

CHANGES

IN BCG

VACCINE

STRAINS

"l

I00 80

40

'°io

F

LI

LII

LEI

[

L~

Fig. 3. T h e c o m p a r i s o n o f i m n a t m i ~ t i o n p o t e n c y , lipid c o n t e n t a n d nuxnber o f Lntact ceils in P r a g u e B C G s ~ cultivated o n S H m e d i u m before a n d after r e p e a t e d lyophilizatlon, K e y as in Fig. 2.

rise and then a gradual decrease in this value to the original l e v e l V,rith repeatedly freeze-dried lots the decrease in the percentage o f intact cells v.'~q far steeper. N o n freeze-dried lots did n o t show any substantial variation in intact mycobacteria percentage hu the course o f the experhnent (Fig. 3). Strain B C G 725 SAS behaved quite differently u n d e r i d e n t i ~ expetqanental conditions. After freeze-drying the zLng poten~¢ increased b u t n o t s and the percentage of h'~tact e l l s decreased, this decrease being significant. T h e inverse proportion o f these two parameters was thus r e d e d in both strains (725 S H and 725 SAS), although their values were reversed. T o t a l Iipids, on the other hand, did not change in either o f the exper;~ne~utal variants (one or several freeze-drying runs). I f these tRree properties are compared in the Danish strain and the two Czechoslovak strains 725 S H and 725 SAS (both o f t h e m reoresent the Danish strain modified by different conditions of maintenance) the Danish strain has the highest immunizing potency and lipid content. T h e Czechoslovak S H strain (non-freeze-dried, Former) ~ s p l a y e d a l-~gher Lm_munizing poten~r and lower percentage of lipids thin: strains SAS (Foimer'). T h e s e two properties are thus inversely proportional. I n neither instance, however, does the potency reach the values of the Danish strain. I n both Czechoslovak strains the pe o f intact c e ~ invariably inversely proportional to immunizing potency (Fig. 4). After freeze-dr-fi_ng, the in-umunizing potency o f the Danish s t u n remains above that of both of the Czechoslovak sh-ains. I n c o n ~ t to n o n - f r e e ~ - d r i e d strains, however, it is fly lower in 725 SH than 725 SAS. T h e percentage of intact cells after freezedrying is lower in 725 S H than 725 SAS. T h i s renders the relation bety~'een the percentage of intact cel~, immunizing potency and total lipids in both Czechoslovak strains after freeze-dEcing directly proportional, in c~ntrast to the pre-freeze-drying situation where only the influence of different culture media comes into pl~¢. However, the relation between irrumu.JzLng potency and percentage o f intact cells was inversely proportional in both strains before and after freeze-drying ~ k e (Fig. 5).

DISCUSSION As regards the immunizing potency, the Danish strain u ~ d in this study concomitantly v~th the two Czechoslovak strains has K e n c ~ d among the topmost strains by Jgspersen (1971) Lu his extensive e x p e r L m e n ~ -work..Another reason for our employing 323

J. P R O C H O V A ,

1VI, M A R A ,

H. 1VIOHELSKA,

Z. ~fR AND

J. G A L L I O V f l .

100 S0 0 |00 50 0

0

F

• ' SL

F

SL

F

SL

Fig. 4. T h e influence of Iyopb/Iization o n the irnrnunization potency, lipid c o n t e n t a n d ntumber o f intact cells in Prague B C G strain cultivated on S A S or S H m e d i m a n d in D a n i s h BCG stY. K e y as in Fig. 2. (F, F o y e r ; S L , freeze-drled vaccine.)

150

100 50 0 150

I00 50

0

]Fig. 5. ~ e influence o f oaltivatlon m e d i u m o n t h e ion potency, lipid c o n t e n t a n d n t L m b ~ o f intact c~l~ in Pre~g~ae B e G strain ¢ u h i ~ t e d on S A S o r S H n - t e ~ m a n d in D a n i s h B e G str',~n ~ ) . K e y as in ]Fig. 2. this strain as a standard of c vv~q t h a t b o t h o f t h e C ~ c h o s l o v a k s t r a i n s w e r e d e r i v e d f r o m it. T h u s , a c t u a l l y , t h e y r e p r e s e n t e d t w o v a r i a n t s o f s t r ~ u B C G C o p e n h a g e n i n v e h i c h m o d i f i c a t i o n s h a v e d e v - e l o p e d a8 a r e s u l t o f t h e d i s t i n c t m a i n t e n a n c e c o n d i ~ o n s of ~ch. Strain BCG 725 SH has been d in Sauton's medium ~ein h y d r o l y s a t e , w h i ~ haLs a r i c h c o n t e n t o f d i f f e r e n t a m / n o a c i d s . S a u t o n ' s o r i g i n a l m e d i u m , o n t h e o t h e r h a n d , o n l y c o n t a i n s o n e a m i n o a c i d , asparagine. S t r a i n B C G 725 S A S w a s o a h i v a t e d u p o n S a u t o n ' s p o t a t o , i.e. a s c o m p a r e d ~ 4 t h s t r a i n B C G C o p e n h a g e n , t h e c u l ~ r e m e d i u m w a s o o m s i d e r a b l y enric~ned b y t h e b i o d a e r n i ~ c o m p o n e n t s o f t h e p o t a t o . I t is t h e r e f o r e p r e s u m e d t h a t t h e s e B C G s t r a i n s w e r e ~ l y influenced by the culture media, markedly richer than those in which they were ~ maintained. The immun;~zing poten~ of the BCG vaccines was measured in immuni~tion experim_ents i n w h i c h t h e d i f f e r e n c e b e t w e e n g u i n e a p i g g r o u p s i n ~ c t e d o n l y a n d g r o u p s 324

g~ h~

Plate 1. BCG 725 SH, age 7 days. Cavitation starting in the nuclear re#on.

Plate2. BCG 725 SH, age 7 days, 3reaks in plasma membr~e and cell wall. CytopI~m is being released into he environment.

Plate 3. BCG 727 SAS, age 7 days, Cavitation in the nuclear region and autolysi s.

Plate 4, BCG 725 SAS. age 7 days, Mesosomal appmatus of the cell in lamdlar form.

Plate 5. BCG 725 SAS. age 7 days. M~osomal apparatus of the cell in tubuhr form,

~ g

:;. i ?

Plate 6. BCG 725 SAS, age 7 days. Vacuoles of unusual shapes surrounded by a simple membrane.

\\

~late 7.

¢

.2

.

L.

3CG 725 SAS, age 7 days. Vacuoles of unusual shapes surrounded by a simple membrane. Same as in Fig, I1.

i

°

7

C H A N G E S IN BCG V A C C I N E S T R A I N S i m m u n i ~ d and challenged "was evaluated. T h r e e criteria were available for evaluatg-~g the significance o f intergroup differences =mean spleen weight, Feldman's index (quantitative evaluation o f visceral T B lesions) and m e a n survival time. Since a positive correlation had previously been found between mean spleen weight, FeIdman's index and m e a n su.~ival time, mean spleen weight alone was used for the evaluation. T h i s m e t h o d has been used routinely on a long-term basis in our laborator$. I n order to ensure comparability between individual immunization experiments (Pr,Schov~i et aL, 1971) Prt$chovh introduced evaluation of results in percentages. T h e e~sence of this approach is outlined in the Materials and M e t h o d s section. Since 1964 we have regularly been checking the g potency of the D ~ s h vaccine w h e n routinely testing the Cze~oslova~k vaccine and f o u n d p e r m a n e n t l y high L,mmunming values with m i n o r . Pr-~chov~i et aL (I971) arrived at estimates of 94% arid 83% immunizing potency for the Danish and C ~ c h o s l o v a k vaccines, respectively. Since 1967 ~ e DarAsh BCG s t r ~ n has been maintained by the seed lot method. A s i g n ~ c a n t difference was found between the summary results of immunization experiments p d before and since the introduction of seed lot for r a c i n e p r o d u c t i o n ~ the values before freeze-dry~mg were somewhat higher t h ~ were those after the adoption of this preservation technio2ae. However, it should also be borne in m i n d that prior to its first freeze-drying the Danish s ~ n was cultivated on bile potato ~ d this factor could have exerted some influence on the changes in the immunizing potency and c h e m i s ~ o f the swain. Our routine resqalts over an 8-year period when the lipid, protein and polysaceiharide contents of the Czechoslovak B C G s ~ a i n SH and the Danish B C G strain -sere investigated were published previously (M~ra, gir, Galliovfi & HynEica, 1972). T h e highest s t a b i l i ~ in the contents of these three c o m p o n e n ~ during maintenance in the usual m a n n e r has been displayed by the Da~nish strain. After freeze-drcing, the lipid content d e c r e e d in both the Danish and the Czechoslovak strain, the protein content increased ira the Danish and d e c r e ~ e d in the Czechoslovak strain and the p o l y s a c c h ~ d e content r e m ~ e d stable in both. F r e e . e - d r y i n g also led to a decrease in the content of mycolie acids and waxes C and D. T h i s physical treah~nent had no effect on any o f the o t h e r fatty acids, as h~Lq been demonstrated by ~ t h paper mad gas ehrom (M~ra, glr, Hejdov.~i eg Mfirowf, 1973; Mfira, Julfik & gir, 1974). It was also found in previous experiments, by means of a spedMly devised method o f mycolie acid preparation by thin-layer chrom that freeze-dr)qng changed the ratio of the two main B C G mycolic acids (alpha and beta). T h i s ratio varied between different B C G strains (Michalec, t~d~ra & A d ~ c o v ~ , 1975 ; rvifira et aL, 1974). W h e n a new glycerine batch was u.~d for preparing culture m e d i u m f o r s t ~ in B C G 725 SH in previous experiments, the lipid and protein content increased while the polysaecharide content was si~ifiea.-atly decreased. Presumably, different contents of trace elements in the two glycerine batches m i g h t have been resp~onsible. Pathologically damaged bacteria were appraised according to the criteria proposed by Armstrong & d'Arcy Hart (1971). T h e s e authors worked with monolayer cultaares of peritoneal macrophages infected with .~'. tuberculosis H37Rv, JVL bG~,2s strain and B C G Moreau. T h e multiplicity of infection was far below 1, i.e. only some of the macrophages were infected with single mycobacteria. T h e resul~ were read electronmicro~copically and in parallel, in terrra o f viable cell counts. According to the f i n d i n ~ (total n u m b e r of 325

J. P R O C H O V A , M. ~ R A , bacteria in morphologies), 'p d by

H. I~¢IOHELSKA, Z. ~ I R A N D J. G A L L I O V t .

age cult,.ire as related to live bacteria and comping_son of bacterial ally damaged' bacteria were n o t viable. T h e divergencies & d ' ~ - c y Hart as pathological damage are as follows:

(I) Gross cavitations havLng n o Hint'ring membr-~te and tly affecting the nuclear region o f the cell. ~ e s e cavitations were frequent in tZhe present experim e n t s (Plate 1). (2) n of cytoplasmic contents through breaks in the plasrmx m e m b r a n e (and obviously the cell wall), i.e. autolysis. T h i s defect was also c o m m o n in the present experiments (Plate 2). (3) Lami , often leading to the form_adon of myelin-like figures. T h e y affect the cell wall and the electron-transparent zone surrou_nding it (the outer envelope of the cell). T h e s e forms were never e n ~ u n t e r e d in the present work. (4) Combinations of the above defects. I n the present material the frequent combination o f defects (1) and (2) ;~as d (Plate 3). According to these criteria, counts o f intact, d or dubious cells were made, the last attribute applies to cases where a safe decision was impossible, in partjc-alar because the was outside the section plane. Intact bacterial cture was mainly evaluated after Avakjan, Kac & Pavlova (1972). I t ~ rather complicated, e~speciaIly on account o f mesosomal apparatus polym o r p h y (Plates 4 and 5) and the presence of vacuoles, frequently of v e ~ bizarre shape, enveloped by a simple m e m b r a n e (Plates 6 and 7). T h e s e are considered b y Avakjmn to be e m p t y spaces after lipid L-~clusions, possibly extracted during specimen preparation. Although no standpoint is being taken here about the nat~are of these forms, their copious presence was indeed observed m oaltures of the B C G variants u n d e r study. T h e results obtained i n d i c t e d that, especially as far as strain B C G 72S BH ~ concerned, the Lmmunizing potency o f mycobacteria was directly proportional to the percentage o f intact cells. Hence the strain seems to contain two variants o f bacteria of which the one more resistant to mechanical action possesses a lower, and the more f ~ g i l e one a higher, immunizing potency. A change in the external conditions, in the present ~ e the p h y s i ~ effect of freeze-drying, brings about a selectAon of the variant o f superior resistance and h-fferior g potency. T h i s selection is incomplete, because after further cultivation in the usual m e d i u m and repeated freeze-drying fhe suppressed variant regains its prevalent position. Attention to the danger of mutation arising and selection during the m~intenance of B C G sta-aLns and during frec~-dryi_ng for the seedlot system is drawn ~ t h m u c h e~mphasis by G u l d (1971). T h e present findings support his views. T h e results o b t ~ n e d -with the Danish and the two ovak B C G strains that substantial changes develop after physical action (freeze-drying) only in st~ains being m ~ t a i n e d o n rich culture media. T h e t w o - m u t a n t t h e o ~ explains the dkfficulties encountered in t h e freeze-drylng of strain 725 SH. Although the decision was taken to use the most sparing m a n n e r ofdr)-mg, with all conditions adjusted accordingly (pre-freezing medium, etc.), the F~ of sur~¢ival was invariably and inva~iably the immunizing potency decreased. T h e present sta~dy was concerned with strains whose properties had been influenced by chemical (maintena~nce, m e d i u m m o ~ c a t i o n s ) and physical action (freeze-drying). T h e results .'o~licate that a straLn exposed to a certain kind o f influence (e.g. ~ e m i c a l ) 326

CHANGES

IN B e G

VACCINE STRAINS

reacts to action o f ~ o t h e r type (e.g. physical) i n a different way t h a n does a strain that has n o t b e e n acted u p o n i n t h e f o r m e r (chemical) m ~ e r , a n d v/ce versa. REFERENCES _Armstrong, J. A. & D,Arcy Hart, P. (1971). Response o f cultured macropkages to d4dycobacterium tuberculosis w i t h observafiorm o n fiasion of lysosomes ~ t h pEagosomes, ffou~,a/ of E x p e r l m ~ t a l 1 ~ , 713-740. Avakjan, A. A., Kac, L. ~q[., ~ Pavlova, I. B. (1972). Atlas aruztomii bakterij patogeun~ch alia geloveka i . rvledicina . G ~ d J. (1971). International S y m p o s i m o n B C G Vaccine, F r a ~ f i a r t m 1VIaLu, 1970. Sy~nposium Series in Immunobiological Standardisation, voL 17, pp. 143-146. Basel: Kargen. Jespersen, A. (1971). The Potenoj o f BCG- Vacdnes Determined on Animal's, p. 91. Cop : Statens S e r u m Instieate. l'dfira, 1H., Ga!!iov~, J. & Hejlov~, E. (1971). Wfiv kultiva~nich podmLuek n a chemick6 slo~eni pra~sktho a kodn-eisk~ho B C G ~ e _ n e . Studia Pn~motogica et P h t 31, 5/6, 196-206. rdfira, 1VI., J ~ , J. & ~ir, Z. (1974). Plynovfi chromatografie mastm#ch kyselin ~ z n # c h ~ e n _ f i B C G v r~azn3~cla kaltiva~_ic~h a l y o ~ z a ~ n i c h podminkfich. Studia Pneumologica et Ph ~ , 308-313. M~ra, M., ~ k , g . , Gnlliovfi, J. & Hyn~ica, V. (1972). Probl6my stabilit~y protituberkttl6zni v a k c ~ y . AHE,~/I 4, 100-1 ~ . M~ra, i . , ~ir, Z., Hejdov~, E. & ~t~fro-ca, E. (1973). St~_~diurnm ~ n n~kter#ch lipidick#ch frakci B e G , l ~ e n f a v kultivaEaxich a lyofi3iza~aaieh podmink~ch. Studia Pneumologica et Phtiseolo~ca ~ , 3/4, 254-263. Mie.halec, ~., Mfira, ?eL & Adamcov~i, D. (I975). Quantitative a n d q u ~ t a t i v e thin-layer c h r o m a t o g r a p h y o f mycolic acids in rium tuberculosis vat. bovis-BCG. Journal of Chromatography 104, 4 6 0 4 6 4 . 1VIohels~, H . & Pr~daov~, J. (1972). Comparative s t u d y o f fine m o r p h o l o g y o f t h e Czechoslovak a n d D a n i s h B C G vaccine strain. Journal of Hygiene, Epidemiology, Microbiology and Immunology 16, 328-333. Pnhchov§, J. & Hejdov~, E. (1971). Evaluation o f the protective effect o f t h e Prague B C G strain 725 Lu comparison with t h e D a n i s h strain 1331 (spleen weight h-a ~ainea pigs). m Series o f immuno Standards 17, 251-258. PrfiChov~, J. & ~ir, Z. (1972). C o m p m t i v e assay o f Lmrnunization potencies o f the C z e c h o slovak, D a n i s h a n d Japanese vaccines as a p r e l i w d n ~ step to t h e seed lot system in t h e p r o d u c t i o n o f B C G vaccine in Czechoslovakia. Progress in Immun Standardization S, 442 a,~6. Basel: Karger. Reynolds, E. S. (1963). T h e use o f lead citrate at h i g h p H as an d e c t r o n o p a q u e stain in d e c t r o n m i c r o s ~ p y , ffournal of Cell Biology 17, 208-211. Ryter, A., Kellenberger, E., Birch-_Andea-son, A. & NIaaloe, O. (1958) ~t~dde all microscope 6 h c t r o n i q u e de plasmas c o n t e n ~ t d e l ' a d d e d t s o ~ i b o n u e l t i q u e . I. Lea n u c l t o i d e s des bach&ties en crois~smuce active. Zeitschrift fiTr ZV.aturforschung 13b, 567-605.

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